The nanopore is a molecular scale pore structure fabricated in an insulating membrane that separate solutions on both sides. Individual target molecules passing through the pore characteristically block the pore conductance, resulting in a signature for both target identification and quantization. The nanopore technology provides a unique single-molecule platform for a variety of biotechnological applications, and in particular the next generation DNA sequencing20-23. In miRNA detection, the nanopore sensor uses a programmable DNA probe to generate a target-specific signature signal, and can quantify subpicomolar levels of miRNAs (such as cancer-associated miRNAs), and can distinguish single-nucleotide differences between miRNA family members. This approach is potentially useful for quantitative miRNA detection, the discovery of disease markers and non-invasive early diagnosis of diseases such as cancer.
Previously disclosed nanopore-based multiplex detection systems include nanopore detection of multiple divalent metal ions, which used a chelator engineered in the pore lumen to generate distinct signatures24 and the use of a molecular adapter to discriminate structure-similar compounds, including pharmaceuticals25, enantiomers26, 27 and nucleotides28, 29. Multiple proteins were also detected in the nanopore by attaching a polymer to the recognition groups.30 Different lengths of free polyethylene glycol (PEG) translocating through the β-barrel of a nanopore can be separated according to the nanopore conductance levels33. It has also been reported that DNA labeled with a polypeptide can generate signatures when trapped in the nanopore31. For the tagging method, the chemical modification of DNA with a peptide tag not only slows the DNA translocation speed, but can generate signatures to facilitate the sensing of individual DNA strands31, including single-base mutations32. The nanopore also functions as a single-molecule mass spectrometry to analyze different sized poly(ethylene glycol) (PEG) polymers translocating through the pore33 and recently, nanopores have been shown to discriminate four bases by detecting four different sized PEG tags released from 5′-phosphate-modified nucleotides34. By chemical modification with a crown tag, individual DNA abase sites can be detected during electrophoretic translocation through the nanopore35. However, the development of high throughput nanopore arrays, in which each pore measures one oligonucleotide, remains a challenge.
One of the challenges to the clinical application of nanopore detection is that specific disease diagnostics usually requires accurate detection of a biomarker panel that consists of multiple miRNAs, rather than a single miRNA species. For example, the combination of three miRNA biomarkers miR-155, miR-182 and miR-197 can increase lung cancer discrimination power to a sensitivity of 81% and specificity of 87%4. This requires simultaneous detection of multiple miRNAs.
Therefore, there is a need to provide a new oligonucleotide detection method based on nano-scale pore structure with improved sensitivity, speedy process, and cost efficiency, as well as providing for multiplex detection.